Sentinel lymph nodes (SLNs) are the first lymph nodes to drain wastes originated from cancerous tissue. There is a need for an in vivo imaging method that can image the intact SLN to further our understanding of its normal as well as abnormal functions. We report the use of ultrahigh sensitive optical microangiography (UHS-OMAG) to image functional microvascular and lymphatic vessel networks that innervate the intact lymph node in mice in vivo. The promising results show a potential role of UHS-OMAG in the future understanding and diagnosis of the SLN involvement in cancer development.

Near-infrared laser-based microsurgery is promising for noninvasive cancer treatment. To make it a safe technique, a therapeutic process should be controllable and energy efficient, which requires the cancer cells to be identifiable and observable. In this work, for the first time we use a miniaturized nonlinear optical endomicroscope to achieve microtreatment of cancer cells labeled with gold nanorods. Due to the high two-photon-excited photoluminescence of gold nanorods, HeLa cells inside a tissue phantom up to 250 µm deep can be imaged by the nonlinear optical endomicroscope. This facilitates microsurgery of selected cancer cells by inducing instant damage through the necrosis process, or by stopping cell proliferation through the apoptosis process. The results indicate that a combination of nonlinear endomicroscopy with gold nanoparticles is potentially viable for minimally invasive cancer treatment.

Fluorescence molecular tomography (FMT) systems coupled to conventional imaging modalities such as magnetic resonance imaging (MRI) and computed tomography provide unique opportunities to combine data sets and improve image quality and content. Yet, the ideal approach to combine these complementary data is still not obvious. This preclinical study compares several methods for incorporating MRI spatial prior information into FMT imaging algorithms in the context of in vivo tissue diagnosis. Populations of mice inoculated with brain tumors that expressed either high or low levels of epidermal growth factor receptor (EGFR) were imaged using an EGF-bound near-infrared dye and a spectrometer-based MRI-FMT scanner. All data were spectrally unmixed to extract the dye fluorescence from the tissue autofluorescence. Methods to combine the two data sets were compared using student's t-tests and receiver operating characteristic analysis. Bulk fluorescence measurements that made up the optical imaging data set were also considered in the comparison. While most techniques were able to distinguish EGFR(+) tumors from EGFR(-) tumors and control animals, with area-under-the-curve values=1, only a handful were able to distinguish EGFR(-) tumors from controls. Bulk fluorescence spectroscopy techniques performed as well as most imaging techniques, suggesting that complex imaging algorithms may be unnecessary to diagnose EGFR status in these tissue volumes.

Three-dimensional tumor models have emerged as valuable in vitro research tools, though the power of such systems as quantitative reporters of tumor growth and treatment response has not been adequately explored. We introduce an approach combining a 3-D model of disseminated ovarian cancer with high-throughput processing of image data for quantification of growth characteristics and cytotoxic response. We developed custom MATLAB routines to analyze longitudinally acquired dark-field microscopy images containing thousands of 3-D nodules. These data reveal a reproducible bimodal log-normal size distribution. Growth behavior is driven by migration and assembly, causing an exponential decay in spatial density concomitant with increasing mean size. At day 10, cultures are treated with either carboplatin or photodynamic therapy (PDT). We quantify size-dependent cytotoxic response for each treatment on a nodule by nodule basis using automated segmentation combined with ratiometric batch-processing of calcein and ethidium bromide fluorescence intensity data (indicating live and dead cells, respectively). Both treatments reduce viability, though carboplatin leaves micronodules largely structurally intact with a size distribution similar to untreated cultures. In contrast, PDT treatment disrupts micronodular structure, causing punctate regions of toxicity, shifting the distribution toward smaller sizes, and potentially increasing vulnerability to subsequent chemotherapeutic treatment.

Cardiolipin is a unique phospholipid of the mitochondrial inner membrane. Its peroxidation correlates with release of cytochrome c and induction of apoptosis. The phthalocyanine photosensitizer Pc 4 binds preferentially to the mitochondria and endoplasmic reticulum. Earlier Förster resonance energy transfer studies showed colocalization of Pc 4 and cardiolipin, which suggests cardiolipin as a target of photodynamic therapy (PDT) with Pc 4. Using liposomes as membrane models, we find that Pc 4 binds to cardiolipin-containing liposomes similarly to those that do not contain cardiolipin. Pc 4 binding is also studied in MCF-7c3 cells and those whose cardiolipin content was reduced by treatment with palmitate. Decreased levels of cardiolipin are quantified by thin-layer chromatography. The similar level of binding of Pc 4 to cells, irrespective of palmitate treatment, supports the lack of specificity of Pc 4 binding. Thus, factors other than cardiolipin are likely responsible for the preferential localization of Pc 4 in mitochondria. Nonetheless, cardiolipin within liposomes is readily oxidized by Pc 4 and light, yielding apparently mono- and dihydroperoxidized cardiolipin. If similar products result from exposure of cells to Pc 4-PDT, they could be part of the early events leading to apoptosis following Pc 4-PDT.

Fluorescent probes are frequently employed for the detection of different reactive oxygen and nitrogen species formed during the irradiation of photosensitized cells and tissues. Investigators often interpret the results in terms of information provided with the different probes without examining specificity or determinants of fluorogenic reactions. We examine five fluorescent probes in a cell-free system: reduced 2,7-dichlorofluorescein, dihydroethidine, dihydrorhodamine, 3-(p aminophenyl) fluorescein (APF), and 4,5-diaminofluorescein. Of these, only APF demonstrates a high degree of specificity for a single reactive species. There is a substantial influence of peroxidase activity on all fluorogenic interactions. The fluorescence of the photosensitizing agent also must be taken into account in evaluating results.

Fluorescence imaging for detection of non-muscle-invasive bladder cancer is based on the selective production and accumulation of fluorescing porphyrins-mainly, protoporphyrin IX-in cancerous tissues after the instillation of Hexvix®. Although the sensitivity of this procedure is very good, its specificity is somewhat limited due to fluorescence false-positive sites. Consequently, magnification cystoscopy has been investigated in order to discriminate false from true fluorescence positive findings. Both white-light and fluorescence modes are possible with the magnification cystoscope, allowing observation of the bladder wall with magnification ranging between 30× for standard observation and 650×. The optical zooming setup allows adjusting the magnification continuously in situ. In the high-magnification (HM) regime, the smallest diameter of the field of view is 600 microns and the resolution is 2.5 microns when in contact with the bladder wall. With this cystoscope, we characterized the superficial vascularization of the fluorescing sites in order to discriminate cancerous from noncancerous tissues. This procedure allowed us to establish a classification based on observed vascular patterns. Seventy-two patients subject to Hexvix® fluorescence cystoscopy were included in the study. Comparison of HM cystoscopy classification with histopathology results confirmed 32/33 (97%) cancerous biopsies and rejected 17/20 (85%) noncancerous lesions.

Topical 5-aminolevulinic acid (ALA) is widely used in photodynamic therapy (PDT) of actinic keratoses (AK), a type of premalignant skin lesion. However, the optimal time between ALA application and exposure to light has not been carefully investigated. Our objective is to study the kinetics of protoporphyrin IX (PpIX) accumulation in AK after short contact ALA and relate this to erythemal responses. Using a noninvasive dosimeter, PpIX fluorescence measurements (5 replicates) were taken at 20-min intervals for 2 h following ALA application, in 63 AK in 20 patients. Data were analyzed for maximal fluorescent signal obtained, kinetic slope, and changes in erythema. Our results show that PpIX accumulation was linear over time, becoming statistically higher than background in 48% of all lesions by 20 min, 92% of lesions by 1 h, and 100% of lesions by 2 h. PpIX accumulation was roughly correlated with changes in lesional erythema post-PDT. We conclude that significant amounts of PpIX are produced in all AK lesions by 2 h. The linear kinetics of accumulation suggest that shorter ALA application times may be efficacious in many patients. Noninvasive fluorescence monitoring of PpIX may be useful to delineate areas of high PpIX accumulation within precancerous areas of the skin.

We describe three lessons learned about how tissue optics affect the dosimetry of red to near-infrared treatment light during PDT, based on working with Dr. Tayyaba Hasan. Lesson 1-The optical fluence rate near the tissue surface exceeds the delivered irradiance (E). A broad beam penetrates into tissue to a depth (z) as =Eke−µz, with an attenuation constant µ and a backscatter term k. In tissues, k is typically in the range 3-5, and 1/µ equals , the 1/e optical penetration depth. Lesson 2-Edge losses at the periphery of a uniform treatment beam extend about 3 from the beam edge. If the beam diameter exceeds 6, then there is a central zone of uniform fluence rate in the tissue. Lesson 3-The depth of treatment is linearly proportional to (and the melanin content of pigmented epidermis in skin) while proportional to the logarithm of all other factors, such as irradiance, exposure time, or the photosensitizer properties (concentration, extinction coefficient, quantum yield for oxidizing species). The lessons illustrate how tissue optics play a dominant role in specifying the treatment zone during PDT.

Wave aberrations of isolated ex vivo porcine crystalline lenses were measured by using a point-diffraction interferometer. This method allowed us to gain greater insight into the detailed aberration structure of eye lenses showing systematic presence of some dominant aberrations. In order of significance, astigmatism together with spherical aberration, coma, and trefoil are the main aberrations present in all lenses. We found a high correlation between the axis of both astigmatism and trefoil with the Y-shaped suture planes of the lens, revealing a subtle relationship between the induced aberrations and the histological features.

Molecular interactions of optical clearing agents were investigated using a combination of molecular dynamics (MD) simulations and optical spectroscopy. For a series of sugar alcohols with low to high optical clearing potential, Raman spectroscopy and integrating sphere measurements were used to quantitatively characterize tissue water loss and reduction in light scattering following agent exposures. The rate of tissue water loss was found to correlate with agent optical clearing potential, but equivalent tissue optical clearing was measured in native and fixed tissue in vitro, given long-enough exposure times to the polyol series. MD simulations showed that the rate of tissue optical clearing correlated with the preferential formation of hydrogen bond bridges between agent and collagen. Hydrogen bond bridge formation disrupts the collagen hydration layer and facilitates replacement by a chemical agent to homogenize tissue refractive index. However, the reduction in tissue light scattering did not correlate with the agent index of refraction. Our results suggest that a necessary property of optical clearing agents is hyperosmolarity to tissue, but that the most effective agents with the highest rates of optical clearing are a subset with the highest collagen solubilities.

A double-integrating-sphere system was used to measure the diffuse transmittance, diffuse reflectance, and collimated transmittance of cartilage and polyacrylamide hydrogel samples as a function of temperature under 1560-nm laser heating conditions. The dynamic behavior of the absorption and scattering coefficients and scattering anisotropy of the biomaterials was calculated by the inverse Monte Carlo method. The absorption coefficient of the cartilage and hydrogel samples proved to be linear in temperature. Raising the temperature of the cartilage samples to 80°C caused their absorption coefficient to decrease by some 25%. The temperature-induced change of the absorption spectrum of the interstitial water was found to be responsible for the clarification of the cartilage tissue observed to occur under 1560-nm laser heating conditions. The temperature field produced in the tissue by the laser energy deposited therein was calculated using a bioheat transfer equation with temperature-dependent parameters. The calculation results demonstrated that the temperature-induced changes of the optical parameters of biological tissues should be taken into account to make their 1560-nm laser treatment effective and safe.

It is known that the fibrous structure of muscle causes light scattering. This phenomenon occurs due to the refractive index discontinuities located between muscle fibers and interstitial fluid. To study the possibility of reducing light scattering inside muscle, we consider its spectral transmittance evolution during an immersion treatment with an optical clearing solution containing ethanol, glycerol, and distilled water. Our methodology consists of registering spectral transmittance of muscle samples while immersed in that solution. With the spectral data collected, we represent the transmittance evolution for some wavelengths during the treatment applied. Additionally, we study the variations that the treatment has caused on the samples regarding tissue refractive index and mass. By analyzing microscopic photographs of tissue cross section, we can also verify changes in the internal arrangement of muscle fibers caused by the immersion treatment. Due to a mathematical model that we develop, we can explain the variations observed in the studied parameters and estimate the amount of optical clearing agent that has diffused into the tissue samples during the immersion treatment. At the end of the study, we observe and explain the improvement in tissue spectral transmittance, which is approximately 65% after 20 min.

Plasma osmolarity influences the volume and shape of red blood cells (RBCs). The volume change is inversely related to the hemoglobin concentration and as a consequence to the complex refractive index within the cell. These morphological changes can be linked to changes in the optical behavior of the cells. The optical parameters, absorption coefficient µa, scattering coefficient µs, and effective scattering phase function of red blood cells are investigated in dependence on osmolarity in the spectral range from 250 to 1100 nm. Integrating sphere measurements of light transmittance and reflectance in combination with inverse Monte-Carlo simulations are carried out for osmolarities from 225 to 400 mosmol/L. Osmolarity changes have a significant influence on the optical parameters, which can in part be explained by changes in the complex refractive index, cell shape, and cell volume. Spherical forms of RBCs induced by low osmolarity show reduced scattering effects compared to the normal RBC biconcave disk shape. Spinocytes, which are crenated erythrocytes induced by high osmolarity, show the highest scattering effects. Even only a 10% change in osmolarity has a drastic influence on the optical parameters, which appears to be of the same order as for 10% hematocrit and oxygen saturation changes.

A linear coherent superposition method for estimating the plane wave far-field scattering pattern from multiple biological cells computed by the finite-difference time-domain (FDTD) method is presented. The method enables the FDTD simulation results of scattering from a small number of complex scatterers, such as biological cells, to be used to estimate the far-field pattern from a large group of those same scatterers. The superposition method can be used to reduce the computational cost of FDTD simulations by enabling a single large scattering problem to be broken into smaller problems with more practical computational requirements. It is found that the method works best in cases where there is little multiple scattering interaction between adjacent cells, so the far-field pattern of multicell geometry can simply be calculated as a phase-adjusted linear superposition of the scattering from individual cells. A strategy is also presented for choosing the minimum number of cells in cases with significant multiple scattering interactions between cells.

Ultrasound-modulated optical tomography (UOT) is based on "tagging" light in turbid media with focused ultrasound. In comparison to diffuse optical imaging, UOT can potentially offer a better spatial resolution. The existing Monte Carlo (MC) model for simulating ultrasound-modulated light is central processing unit (CPU) based and has been employed in several UOT related studies. We reimplemented the MC model with a graphics processing unit [(GPU), Nvidia GeForce 9800] that can execute the algorithm up to 125 times faster than its CPU (Intel Core Quad) counterpart for a particular set of optical and acoustic parameters. We also show that the incorporation of ultrasound propagation in photon migration modeling increases the computational time considerably, by a factor of at least 6, in one case, even with a GPU. With slight adjustment to the code, MC simulations were also performed to demonstrate the effect of ultrasonic modulation on the speckle pattern generated by the light model (available as animation). This was computed in 4 s with our GPU implementation as compared to 290 s using the CPU.

The absorption of filter substances in sunscreens, reducing the incident ultraviolet (UV) radiation, is the basis for the protecting ability of such formulations. The erythema-correlated sun protection factor (SPF), depending mainly on the intensity of the UVB radiation, is the common value to quantify the efficacy of the formulations avoiding sunburn. An ex vivo method combining tape stripping and optical spectroscopy is applied to measure the absorption of sunscreens in the entire UV spectral range. The obtained relations between the short-wavelength UV (UVB) absorption and the SPF confirm a clear influence of the long-wavelength UV (UVA) absorption on the SPF values. The data reflect the historical development of the relation of the concentration of UVB and UVA filters in sunscreens and points to the influence of additional ingredients, e.g., antioxidants and cell-protecting agents on the efficacy of the products.

Near-infrared laser (785-nm)-excited Raman spectra from a red blood cell, optically trapped using the same laser beam, show significant changes as a function of trapping duration even at trapping power level of a few milliwatts. These changes in the Raman spectra and the bright-field images of the trapped cell, which show a gradual accumulation of the cell mass at the trap focus, suggest photoinduced aggregation of intracellular heme. The possible role of photoinduced protein denaturation and hemichrome formation in the observed aggregation of heme is discussed.

Birefringent media, like biological tissues, are usually assumed to be uniaxial. For biological tissues, the influence of linear birefringence on the scattering phase function is assumed to be neglectable. In order to examine this, a numerical study of the influence of linear birefringence on the scattering phase function and the resulting backscattering Mueller matrices was performed. It is assumed that the media consist of spherical scattering particles embedded in a nonabsorbing medium, which allows us to employ the Lorenz-Mie theory. In the Monte Carlo framework, the influence of linear birefringence on the components of the electric field vector is captured through the Jones N-matrix formalism. The Lorenz-Mie theory indicates that a given linear birefringence value n has a bigger impact on the scattering phase function for large particles. This conclusion is further supported by Monte Carlo simulations, where the phase function was calculated based on the refractive index once in the ordinary direction and once in the extraordinary one. For large particles, comparisons of the resulting backscattering Mueller matrices show significant differences even for small n values.

The feasibility of air-coupled ultrasound transducers to detect laser-induced ultrasound from artificial blood vessels embedded in an optically scattering phantom is demonstrated. These air-coupled transducers allow new applications in biomedical photoacoustic imaging where contact with tissue is not preferred. One promising application of such transducers is the addition of photoacoustic imaging to the regular x-ray mammographic screening procedure.

Optical nerve stimulation using infrared laser radiation has recently been developed as a potential alternative to electrical nerve stimulation. However, recent studies have focused primarily on pulsed delivery of the laser radiation and at relatively low pulse rates. The objective of this study is to demonstrate faster optical stimulation of the prostate cavernous nerves using continuous-wave (cw) infrared laser radiation for potential diagnostic applications. A thulium fiber laser (=1870 nm) is used for noncontact optical stimulation of the rat prostate cavernous nerves in vivo. Optical nerve stimulation, as measured by an intracavernous pressure (ICP) response in the penis, is achieved with the laser operating in either cw mode, or with a 5-ms pulse duration at 10, 20, 30, 40, 50, and 100 Hz. Successful optical stimulation is observed to be primarily dependent on a threshold nerve temperature (42 to 45 °C), rather than an incident fluence, as previously reported. cw optical nerve stimulation provides a significantly faster ICP response time using a lower power (and also less expensive) laser than pulsed stimulation. cw optical nerve stimulation may therefore represent an alternative mode of stimulation for intraoperative diagnostic applications where a rapid response is critical, such as identification of the cavernous nerves during prostate cancer surgery.

An estimated 1.3 million people in the United States suffer from rheumatoid arthritis (RA). RA causes profound changes in the synovial membrane of joints, and without early diagnosis and intervention, progresses to permanent alterations in joint structure and function. The purpose of this study is to determine if nonlinear optical microscopy (NLOM) can utilize the natural intrinsic fluorescence properties of tissue to generate images that would allow visualization of the structural and cellular composition of fresh, unfixed normal and pathologic synovial tissue. NLOM is performed on rabbit knee joint synovial samples using 730- and 800-nm excitation wavelengths. Less than 30 mW of excitation power delivered with a 40×, 0.8-NA water immersion objective is sufficient for the visualization of synovial structures to a maximum depth of 70 µm without tissue damage. NLOM imaging of normal and pathologic synovial tissue reveals the cellular structure, synoviocytes, adipocytes, collagen, vascular structures, and differential characteristics of inflammatory infiltrates without requiring tissue processing or staining. Further study to evaluate the ability of NLOM to assess the characteristics of pathologic synovial tissue and its potential role for the management of disease is warranted.

A method is presented to select the optimal wavelengths for multispectral optical topography, which not only gives good separation between chromophores, absorption, and scattering, but also minimizes the differences between interrogated volumes. This method uses the sum of squared differences to compare photon measurement density functions, which were generated for wavelengths in the near-infrared (NIR) range for a suitable model of tissue optical properties. It is found that including this condition significantly influences the range of optimal wavelengths. However, for the adult human head, the differences between interrogated volumes at NIR wavelengths are very small and image reconstruction is only slightly improved when measurements with overlapping sensitivities are used.

The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ~2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01±0.10 ns) or Rab7 (2.11±0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78±0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09±0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

Corneal polarimetry measurement has been the object of several papers. The results of techniques like polarization-sensitive optical coherence tomography (PS-OCT), scanning laser polarimetry, or polarization microscopy are contradictory. Some studies propose a biaxial-like birefringence pattern, while others postulate that birefringence grows at corneal periphery. Several theoretical approaches were proposed for the interpretation of these measurements, but they usually lack accuracy and an adequate consideration of the nonnormal incidence on the tissue. We analyze corneal polarization effects measured by PS-OCT. In vivo and in vitro PS-OCT images of the human cornea are acquired. PS-OCT measurements are apparently not in agreement with the biaxial-like birefringence pattern. We present a polarimetric model of the human cornea based on the extended Jones matrix formalism applied to multilayered systems. We also apply the Poincaré equivalence theorem to extract optic axis orientation and birefringence. The results show that for a fibrils orientation pattern composed by alternating circular and radial fibrils, the birefringence is biaxial-like at the corneal center, and there is an almost circularly symmetric high-birefringence area at corneal periphery. The model could be useful for diagnosis of corneal diseases or corneal compensation in retinal polarimetric imaging.

Hearing loss can mean severe impairment to the quality of life. However, the biomechanical mechanisms of how the hearing organ, i.e., the organ of Corti (OC), responds to sound are still elusive, largely because there is currently no means available to image the 3-D motion characteristics of the OC. We present a novel use of the phase-sensitive spectral domain optical coherence tomography (PSOCT) to characterize the motion of cellular compartments within the OC at a subnanometer scale. The PSOCT system operates at 1310 nm with a spatial resolution of ~16 µm and an imaging speed of 47,000 A-lines/s. The phase changes of the spectral interferograms induced by the localized tissue motion are used to quantify the vibration magnitude. Fourier transform analysis of the phase changes improves the system sensitivity to sense minute vibrations smaller than 1 nm. We demonstrate that the PSOCT system is feasible to image the meaningful vibration of cellular compartments within the OC with an unprecedented sensitivity down to ~0.5 Å.

Micrometer scale resolution full-field optical coherence tomography (FF-OCT) is developed for imaging human graft corneas. Three-dimensional (3-D) images with ultrahigh resolution (respectively, 1 and 1.5 µm in the axial and transverse directions), comparable to traditional histological sections, are obtained allowing the visualization of the cells and the precise structure of the different layers that compose the tissue. The sensitivity of our device enables imaging the entire thickness of the cornea, even in edematous corneas more than 800 µm thick. Furthermore, we provide tomographic 3-D images of laser incisions inside the tissue at various depths without slicing the studied corneas. The effects of laser ablations can be observed, along various optical sections, directly in the bulk of the sample with high accuracy, providing information on the interface quality and also imaging tiny changes of the tissue structure. FF-OCT appears to be a powerful tool for subcellular imaging of the corneal structure and pathologies on the entire thickness of the tissue as well as interface quality and changes in the collagen structure due to laser incisions on ex vivo human cornea.

Monitoring liver fibrosis progression by liver biopsy is important for certain treatment decisions, but repeated biopsy is invasive. We envision redefinition or elimination of liver biopsy with surface scanning of the liver with minimally invasive optical methods. This would be possible only if the information contained on or near liver surfaces accurately reflects the liver fibrosis progression in the liver interior. In our study, we acquired the second-harmonic generation and two-photon excitation fluorescence microscopy images of liver tissues from bile duct-ligated rat model of liver fibrosis. We extracted morphology-based features, such as total collagen, collagen in bile duct areas, bile duct proliferation, and areas occupied by remnant hepatocytes, and defined the capsule and subcapsular regions on the liver surface based on image analysis of features. We discovered a strong correlation between the liver fibrosis progression on the anterior surface and interior in both liver lobes, where biopsy is typically obtained. The posterior surface exhibits less correlation with the rest of the liver. Therefore, scanning the anterior liver surface would obtain similar information to that obtained from biopsy for monitoring liver fibrosis progression.

Both photoacoustic imaging and power Doppler ultrasound are capable of producing images of the vasculature of living subjects, however, the contrast mechanisms of the two modalities are very different. We present a quantitative and objective comparison of the two methods using phantom data, highlighting relative merits and shortcomings. An imaging system for combined photoacoustic and high-frequency power Doppler ultrasound microscopy is presented. This system uses a swept-scan 25-MHz ultrasound transducer with confocal dark-field laser illumination optics. A pulse-sequencer enables ultrasonic and laser pulses to be interlaced so that photoacoustic and power Doppler ultrasound images can be coregistered. Experiments are performed on flow phantoms with various combinations of vessel size, flow velocity, and optical wavelength. For the task of blood volume detection, power Doppler is seen to be advantageous for large vessels and high flow speeds. For small vessels with low flow speeds, photoacoustic imaging is seen to be more effective than power Doppler at the detection of blood as quantified by receiver operating characteristic analysis. A combination of the two modes could provide improved estimates of fractional blood volume in comparison with either mode used alone.

Fluorescence imaging in diffusive media is an emerging imaging modality for medical applications that uses injected fluorescent markers that bind to specific targets, e.g., carcinoma. The region of interest is illuminated with near-IR light and the emitted back fluorescence is analyzed to localize the fluorescence sources. To investigate a thick medium, as the fluorescence signal decreases with the light travel distance, any disturbing signal, such as biological tissues intrinsic fluorescence (called autofluorescence) is a limiting factor. Several specific markers may also be simultaneously injected to bind to different molecules, and one may want to isolate each specific fluorescent signal from the others. To remove the unwanted fluorescence contributions or separate different specific markers, a spectroscopic approach is explored. The nonnegative matrix factorization (NMF) is the blind positive source separation method we chose. We run an original regularized NMF algorithm we developed on experimental data, and successfully obtain separated in vivo fluorescence spectra.

Raman tweezers and quantitative differential interference contrast (DIC) microscopy are combined to monitor the dynamic germination of individual bacterial spores of Bacillus species, as well as the heterogeneity in this process. The DIC bias phase is set properly such that the brightness of DIC images of individual spores is proportional to the dipicolinic acid (DPA) level of the spores, and an algorithm is developed to retrieve the phase image of an individual spore from its DIC image. We find that during germination, the rapid drop in both the intensity of the original DIC image and the intensity of the reconstructed phase image precisely corresponds to the release of all DPA from that spore. The summed pixel intensity of the DIC image of individual spores adhered on a microscope coverslip is not sensitive to the drift of the slide in both horizontal and vertical directions, which facilitates observation of the germination of thousands of individual spores for long periods of time. A motorized stage and synchronized image acquisition system is further developed to effectively expand the field of view of the DIC imaging. This quantitative DIC technique is used to track the germination of hundreds or thousands of individual spores simultaneously.

Single spectrometer-based complex conjugate artifact removal methods are evaluated for in vivo imaging with complementary metal-oxide semiconductor line scan camera based high-speed Fourier-domain optical coherence tomography (FD-OCT) at 100,000 axial scans per second. Performance of three different phase-shifting methods with the same OCT engine is evaluated using modified data acquisition schemes, depending on the requirements of each phase-shifting technique. The suppression ratio of complex conjugate artifact images using a paperboard is assessed for all tested methods. Several other characteristics, including a list of additional hardware requirements (beyond standard FD-OCT components) and data acquisition schemes for each of the methods is presented. In vivo full-range images of human fingerpad and nail are shown and compared with standard FD-OCT images. Additionally, a complex-conjugate-free human retinal volume acquired at the speed of 100,000 A-scans/s is presented.

A method for three-dimensional motion analysis designed for live cell imaging by fluorescence confocal microscopy is described. The approach is based on optical flow computation and takes into account brightness variations in the image scene that are not due to motion, such as photobleaching or fluorescence variations that may reflect changes in cellular physiology. The 3-D optical flow algorithm allowed almost perfect motion estimation on noise-free artificial sequences, and performed with a relative error of <10% on noisy images typical of real experiments. The method was applied to a series of 3-D confocal image stacks from an in vitro preparation of the guinea pig cochlea. The complex motions caused by slow pressure changes in the cochlear compartments were quantified. At the surface of the hearing organ, the largest motion component was the transverse one (normal to the surface), but significant radial and longitudinal displacements were also present. The outer hair cell displayed larger radial motion at their basolateral membrane than at their apical surface. These movements reflect mechanical interactions between different cellular structures, which may be important for communicating sound-evoked vibrations to the sensory cells. A better understanding of these interactions is important for testing realistic models of cochlear mechanics.

In biological applications of fluorescence lifetime imaging, low signals from samples can be a challenge, causing poor lifetime precision. We demonstrate how optimal signal gating (a method applied to the temporal dimension of a lifetime image) and novel total variation denoising models (a method applied to the spatial dimension of a lifetime image) can be used in time-domain fluorescence lifetime imaging microscopy (FLIM) to improve lifetime precision. In time-gated FLIM, notable fourfold precision improvements were observed in a low-light example. This approach can be employed to improve FLIM data while minimizing sample light exposure and increasing imaging speed.

We present a new method for the quantitative estimation of blood flow velocity, based on the use of the Radon transform. The specific application is for measurement of blood flow velocity in the retina. Our modified fundus camera uses illumination from a green LED and captures imagery with a high-speed CCD camera. The basic theory is presented, and typical results are shown for an in vitro flow model using blood in a capillary tube. Subsequently, representative results are shown for representative fundus imagery. This approach provides absolute velocity and flow direction along the vessel centerline or any lateral displacement therefrom. We also provide an error analysis allowing estimation of confidence intervals for the estimated velocity.

A technique called surface plasmon resonance digital holographic microscopy (SPRDHM) for optical imaging of cell membranes is proposed. The intensity and phase distributions of the reflected light that is modulated by the cell membrane in surface plasmon resonance can be simultaneously obtained. The imaging principle and capability are theoretically analyzed and demonstrated by experiments. In addition, the technique is compared with total internal reflection digital holographic microscopy (TIRDHM) in theory and experiment, respectively. The results show that the SPRDHM technique is better in spatial resolution and phase sensitivity than the TIRDHM technique for imaging of cell membranes

Pulse-modulated second harmonic imaging microscopes (PM-SHIMs) exhibit improved signal-to-noise ratio (SNR) over conventional SHIMs on sensitive imaging and quantification of weak collagen signals inside tissues. We quantify the spatial distribution of sparse collagen inside a xenograft model of human acute myeloid leukemia (AML) tumor specimens treated with a new drug against receptor tyrosine kinase (ABT-869), and observe a significant increase in collagen area percentage, collagen fiber length, fiber width, and fiber number after chemotherapy. This finding reveals new insights into tumor responses to chemotherapy and suggests caution in developing new drugs and therapeutic regimens against cancers.

Optical-resolution photoacoustic microscopy (OR-PAM) is a novel imaging technology for visualizing optically absorbing superficial structures in vivo with lateral spatial resolution determined by optical focusing rather than acoustic detection. Since scanning of the illumination spot is required, OR-PAM imaging speed is limited by both scanning speed and laser pulse repetition rate. Unfortunately, lasers with high repetition rates and suitable pulse durations and energies are not widely available and can be cost-prohibitive and bulky. We are developing compact, passively Q-switched fiber and microchip laser sources for this application. The properties of these lasers are discussed, and pulse repetition rates up to 100 kHz are demonstrated. OR-PAM imaging was conducted using a previously developed photoacoustic probe, which enabled flexible scanning of the focused output of the lasers. Phantom studies demonstrate the ability to image with lateral spatial resolution of 7±2 µm with the microchip laser system and 15±5 µm with the fiber laser system. We believe that the high pulse repetition rates and the potentially compact and fiber-coupled nature of these lasers will prove important for clinical imaging applications where real-time imaging performance is essential.

Dermal fibroblasts are responsible for the generation of mechanical forces within their surrounding extracellular matrix and can be potentially targeted by anti-aging ingredients. Investigation of the modulation of fibroblast contraction by these ingredients requires the implementation of three-dimensional in situ imaging methodologies. We use multiphoton microscopy to visualize unstained engineered dermal tissue by combining second-harmonic generation that reveals specifically fibrillar collagen and two-photon excited fluorescence from endogenous cellular chromophores. We study the fibroblast-induced reorganization of the collagen matrix and quantitatively evaluate the effect of Y-27632, a RhoA-kinase inhibitor, on dermal substitute contraction. We observe that collagen fibrils rearrange around fibroblasts with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA-kinase inhibitor. Moreover, we show that the inhibitory effects are reversible. Our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the extracellular matrix induced by fibroblast contraction or other processes.

Computed virtual chromoendoscopy with flexible spectral imaging color enhancement (FICE) is a new dyeless imaging technique that enhances mucosal and vascular patterns. However, a method for selecting a suitable wavelength for a particular condition has not been established. The aim of this study is to evaluate the color difference method for quality assessment of FICE images of the intrapapillary capillary loop in magnifying endoscopy for esophageal squamous cell carcinoma. The color difference between 60 microvessels and background mucosa observed using the magnifying endoscope was 8.31±2.84 SD under white light and 12.26±3.14 (p=0.0031), 11.70±4.49 (p=0.0106), and 17.49±5.40 (p<0.0001) in FICE modes A, B, and C, respectively. The visibility scores for microvessels observed by medical students were 6.00±1.12 points under white light and 11.1±2.25 (p<0.0001), 8.65±2.06 (p=0.0001), and 12.55±2.56 (p<0.0001) in FICE modes A, B, and C, respectively. Furthermore, the measurement of color difference was correlated with the visibility score assigned by medical students (Pearson's correlation coefficient=0.583, p<0.0001) In conclusion, the color difference method corresponds to human vision and is an appropriate method for evaluation of endoscopic images.

Drosophila melanogaster (fruit fly) is a central organism in biology and is becoming increasingly important in the cardiovascular sciences. Prior work in optical imaging of the D. melanogaster heart has focused on static and dynamic structural anatomy. In the study, it is demonstrated that Doppler optical coherence tomography can quantify dynamic heart wall velocity and hemolymph flow in adult D. melanogaster. Since hemolymph is optically transparent, a novel exogenous contrast technique is demonstrated to increase the backscatter-based intracardiac Doppler flow signal. The results presented here open up new possibilities for functional cardiovascular phenotyping of normal and mutant D. melanogaster.

Accurate tumor excision is crucial in the locoregional treatment of cancer, and for this purpose, surgeons often rely on guide wires or radioactive markers for guidance toward the lesion. Further improvement may be obtained by adding optical guidance to currently used methods, in the form of intra-operative fluorescence imaging. To achieve such a multimodal approach, we have generated markers that can be used in a pre-, intra-, and post-operative setting, based on a cocktail of a dual-emissive inorganic dye, lipids, and pertechnetate. Phantom experiments demonstrate that these seeds can be placed accurately around a surrogate tumor using ultrasound. Three-dimensional bracketing provides delineation of the entire lesion. Combined with the multimodal nature, this provides the opportunity to predetermine the resection margins by validating the placement accuracy using multiple imaging modalities (namely, x ray, MRI, SPECT/CT, and ultrasound). The dual-emissive fluorescent properties of the dye provide the unique opportunity to intra-operatively estimate the depth of the seed in the tissue via multispectral imaging: emission green max=520 nm5 mm penetration versus emission red max=660 nm12 mm penetration. By using particles with different colors, the original geographic orientation of the excised tissue can be determined.

We demonstrate for the first time the application of an endoscopic fluorescence lifetime imaging microscopy (FLIM) system to the intraoperative diagnosis of glioblastoma multiforme (GBM). The clinically compatible FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system with a fiber-bundle (fiber image guide of 0.5 mm diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, and 4 mm field of view) to provide intraoperative access to the surgical field. Experiments conducted in three patients undergoing craniotomy for tumor resection demonstrate that FLIM-derived parameters allow for delineation of tumor from normal cortex. For example, at 460±25-nm wavelength band emission corresponding to NADH/NADPH fluorescence, GBM exhibited a weaker florescence intensity (35% less, p-value <0.05) and a longer lifetime GBM-Amean=1.59±0.24 ns than normal cortex NC-Amean=1.28±0.04 ns (p-value <0.005). Current results demonstrate the potential use of FLIM as a tool for image-guided surgery of brain tumors.

Red blood cells (RBCs) have been found to undergo "programmed cell death," or eryptosis, and understanding this process can provide more information about apoptosis of nucleated cells. Photothermal (PT) response, a label-free photothermal noninvasive technique, is proposed as a tool to monitor the cell death process of living human RBCs upon glucose depletion. Since the physiological status of the dying cells is highly sensitive to photothermal parameters (e.g., thermal diffusivity, absorption, etc.), we applied linear PT response to continuously monitor the death mechanism of RBC when depleted of glucose. The kinetics of the assay where the cell's PT response transforms from linear to nonlinear regime is reported. In addition, quantitative monitoring was performed by extracting the relevant photothermal parameters from the PT response. Twofold increases in thermal diffusivity and size reduction were found in the linear PT response during cell death. Our results reveal that photothermal parameters change earlier than phosphatidylserine externalization (used for fluorescent studies), allowing us to detect the initial stage of eryptosis in a quantitative manner. Hence, the proposed tool, in addition to detection of eryptosis earlier than fluorescence, could also reveal physiological status of the cells through quantitative photothermal parameter extraction.

Laser Doppler flowmetry (LDF) can be used for assessing the microcirculatory perfusion. However, conventional LDF (cLDF) gives only a relative perfusion estimate for an unknown measurement volume, with no information about the blood flow speed distribution. To overcome these limitations, a model-based analysis method for quantitative LDF (qLDF) is proposed. The method uses inverse Monte Carlo technique with an adaptive three-layer skin model. By analyzing the optimal model where measured and simulated LDF spectra detected at two different source-detector separations match, the absolute microcirculatory perfusion for a specified speed region in a predefined volume is determined. qLDF displayed errors <12% when evaluated using simulations of physiologically relevant variations in the layer structure, in the optical properties of static tissue, and in blood absorption. Inhomogeneous models containing small blood vessels, hair, and sweat glands displayed errors <5%. Evaluation models containing single larger blood vessels displayed significant errors but could be dismissed by residual analysis. In vivo measurements using local heat provocation displayed a higher perfusion increase with qLDF than cLDF, due to nonlinear effects in the latter. The qLDF showed that the perfusion increase occurred due to an increased amount of red blood cells with a speed >1 mm/s.

Because cancer is a dreaded disease, a number of techniques such as biomarker evaluation, mammograms, colposcopy, and computed tomography scan are currently employed for early diagnosis. Many of these are specific to a particular site, invasive, and often expensive. Hence, there is a definite need for a simple, generic, noninvasive protocol for cancer detection, comparable to blood and urine tests for diabetes. Our objective is to show the results of a novel study in the diagnosis of several cancer types from the native or intrinsic fluorescence of urine. We use fluorescence emission spectra (FES) and stokes shift spectra (SSS) to analyze the native fluorescence of the first voided urine samples of healthy controls (N=100) and those of cancer patients (N=50) of different etiology. We show that flavoproteins and porphyrins released into urine can act as generic biomarkers of cancer with a specificity of 92%, a sensitivity of 76%, and an overall accuracy of 86.7%. We employ FES and SSS for rapid and cost-effective quantification of certain intrinsic biomarkers in urine for screening and diagnosis of most common cancer types with an overall accuracy of 86.7%.

A primary focus of neurointensive care is monitoring the injured brain to detect harmful events that can impair cerebral blood flow (CBF), resulting in further injury. Since current noninvasive methods used in the clinic can only assess blood flow indirectly, the goal of this research is to develop an optical technique for measuring absolute CBF. A time-resolved near-infrared (TR-NIR) apparatus is built and CBF is determined by a bolus-tracking method using indocyanine green as an intravascular flow tracer. As a first step in the validation of this technique, CBF is measured in newborn piglets to avoid signal contamination from extracerebral tissue. Measurements are acquired under three conditions: normocapnia, hypercapnia, and following carotid occlusion. For comparison, CBF is concurrently measured by a previously developed continuous-wave NIR method. A strong correlation between CBF measurements from the two techniques is revealed with a slope of 0.79±0.06, an intercept of −2.2±2.5 ml/100 g/min, and an R2 of 0.810±0.088. Results demonstrate that TR-NIR can measure CBF with reasonable accuracy and is sensitive to flow changes. The discrepancy between the two methods at higher CBF could be caused by differences in depth sensitivities between continuous-wave and time-resolved measurements.

I use Monte Carlo simulations and phantom measurements to characterize a probe with adjacent optical fibres for diffuse reflectance spectroscopy during stereotactic surgery in the brain. Simulations and measurements have been fitted to a modified Beer-Lambert model for light transport in order to be able to quantify chromophore content based on clinically measured spectra in brain tissue. It was found that it is important to take the impact of the light absorption into account when calculating the apparent optical path length, lp, for the photons in order to get good estimates of the absorption coefficient, µa. The optical path length was found to be well fitted to the equation lp=a+b ln(Is)+c ln(µa)+d ln(Is)ln(µa), where Is is the reflected light intensity for scattering alone (i.e., zero absorption). Although coefficients a-d calculated in this study are specific to the probe used here, the general form of the equation should be applicable to similar probes.

Neovascularization in cervical intraepithelial neoplasia (CIN) is studied because it is the precursor to the third most common female cancer worldwide. Diffuse reflectance from 450-600 nm was collected from 46 patients (76 sites) undergoing colposcopy at Duke University Medical Center. Total hemoglobin, derived using an inverse Monte Carlo model, significantly increased in CIN 2+ (N=12) versus CIN 1 (N=16) and normal tissues (N=48) combined with P<0.004. Immunohistochemistry using monoclonal anti-CD34 was used to quantify microvessel density to validate the increased hemoglobin content. Biopsies from 51 sites were stained, and up to three hot spots per slide were selected for microvessel quantification by two observers. Similar to the optical study results, microvessel density was significantly increased in CIN 2+ (N=16) versus CIN 1 (N=21) and normal tissue (N=14) combined with P<0.007. Total vessel density, however, was not significantly associated with dysplastic grade. Hence, our quantitative optical spectroscopy system is primarily sensitive to dysplastic neovascularization immediately beneath the basement membrane, with minimal confounding from vascularity inherent in the normal stromal environment. This tool could have potential for in vivo applications in screening for cervical cancer, prognostics, and monitoring of antiangiogenic effects in chemoprevention therapies.

We introduce a method for noninvasively measuring muscle contraction in vivo, based on near-infrared diffusing-wave spectroscopy (DWS). The method exploits the information about time-dependent shear motions within the contracting muscle that are contained in the temporal autocorrelation function g(1)(,t) of the multiply scattered light field measured as a function of lag time, , and time after stimulus, t. The analysis of g(1)(,t) measured on the human M. biceps brachii during repetitive electrical stimulation, using optical properties measured with time-resolved reflectance spectroscopy, shows that the tissue dynamics giving rise to the speckle fluctuations can be described by a combination of diffusion and shearing. The evolution of the tissue Cauchy strain e(t) shows a strong correlation with the force, indicating that a significant part of the shear observed with DWS is due to muscle contraction. The evolution of the DWS decay time shows quantitative differences between the M. biceps brachii and the M. gastrocnemius, suggesting that DWS allows to discriminate contraction of fast- and slow-twitch muscle fibers.

A simple optical model of single lymphocytes with smooth and nonsmooth surfaces has been developed for healthy and infected individuals. The model can be used for rapid (in the real-time scale) solution of the inverse light-scattering problem on the basis of optical data measured by label-free flow cytometry. Light scattering patterns have been calculated for the model developed. It has been shown that the smooth and nonsmooth cells can be resolved using the intensities of the sideward- and backward-scattered light. We have found by calculations and validated by the flow cytometer experiments that intensity distributions for the cells of lymphocyte populations can be used as a preliminary signatures of some virus infections. Potential biomedical applications of the findings for label-free flow cytometry detection of individuals infected with viruses of hepatitises B or C and some others viruses are presented.

Early detection of ovarian cancer could greatly increase the likelihood of successful treatment. However, present detection techniques are not very effective, and symptoms are more commonly seen in later stage disease. Amino acids, structural proteins, and enzymatic cofactors have endogenous optical properties influenced by precancerous changes and tumor growth. We present the technical details of an optical spectroscopy system used to quantify these properties. A fiber optic probe excites the surface epithelium (origin of 90% of cases) over 270 to 580 nm and collects fluorescence and reflectance at 300 to 800 nm with four or greater orders of magnitude instrument to background suppression. Up to four sites per ovary are investigated on patients giving consent to oophorectomy and the system's in vivo optical evaluation. Data acquisition is completed within 20 s per site. We illustrate design, selection, and development of the components used in the system. Concerns relating to clinical use, performance, calibration, and quality control are addressed. In the future, spectroscopic data will be compared with histological biopsies from the corresponding tissue sites. If proven effective, this technique can be useful in screening women at high risk of developing ovarian cancer to determine whether oophorectomy is necessary.

Calcium is an important messenger in cells and whose store and diffusion dynamics at the subcellular level remain unclear. By inducing a controlled slow subcellular Ca2+ release through femtosecond laser irradiation in HeLa cells immersed in different media, cytoplasm is identified to be the major intracellular Ca2+ store, with the nucleus being the minor store and the extracellular Ca2+ also contributing to the total cellular Ca2+ level. Furthermore, Ca2+ released in either the cytoplasm or nucleus diffuses into the nucleus or cytoplasm, respectively, at different rates and influences the Ca2+ release in those regions.

The prescribed radiant exposures for photodynamic therapy (PDT) of superficial skin cancers are chosen empirically to maximize the success of the treatment while minimizing adverse reactions for the majority of patients. They do not take into account the wide range of tissue optical properties for human skin, contributing to relatively low treatment success rates. Additionally, treatment times can be unnecessarily long for large treatment areas if the laser power is not sufficient. Both of these concerns can be addressed by the incorporation of an integrating sphere into the irradiation apparatus. The light fluence rate can be increased by as much as 100%, depending on the tissue optical properties. This improvement can be determined in advance of treatment by measuring the reflectance from the tissue through a side port on the integrating sphere, allowing for patient-specific treatment times. The sphere is also effective at improving beam flatness, and reducing the penumbra, creating a more uniform light field. The side port reflectance measurements are also related to the tissue transport albedo, enabling an approximation of the penetration depth, which is useful for real-time light dosimetry.

We conduct a comparative study on the efficiency and cell death pathways of continuous wave (cw) and nanosecond pulsed laser photothermal cancer therapy using gold nanospheres delivered to either the cytoplasm or nucleus of cancer cells. Cytoplasm localization is achieved using arginine-glycine-aspartate peptide modified gold nanospheres, which target integrin receptors on the cell surface and are subsequently internalized by the cells. Nuclear delivery is achieved by conjugating the gold nanospheres with nuclear localization sequence peptides originating from the simian virus. Photothermal experiments show that cell death can be induced with a single pulse of a nanosecond laser more efficiently than with a cw laser. When the cw laser is applied, gold nanospheres localized in the cytoplasm are more effective in inducing cell destruction than gold nanospheres localized at the nucleus. The opposite effect is observed when the nanosecond pulsed laser is used, suggesting that plasmonic field enhancement of the nonlinear absorption processes occurs at high localization of gold nanospheres at the nucleus. Cell death pathways are further investigated via a standard apoptosis kit to show that the cell death mechanisms depend on the type of laser used. While the cw laser induces cell death via apoptosis, the nanosecond pulsed laser leads to cell necrosis. These studies add mechanistic insight to gold nanoparticle-based photothermal therapy of cancer.

The first results from a clinical study for Temoporfin-mediated photodynamic therapy (PDT) of low-grade (T1c) primary prostate cancer using online dosimetry are presented. Dosimetric feedback in real time was applied, for the first time to our knowledge, in interstitial photodynamic therapy. The dosimetry software IDOSE provided dose plans, including optical fiber positions and light doses based on 3-D tissue models generated from ultrasound images. Tissue optical property measurements were obtained using the same fibers used for light delivery. Measurements were taken before, during, and after the treatment session. On the basis of these real-time measured optical properties, the light-dose plan was recalculated. The aim of the treatment was to ablate the entire prostate while minimizing exposure to surrounding organs. The results indicate that online dosimetry based on real-time tissue optical property measurements enabled the light dose to be adapted and optimized. However, histopathological analysis of tissue biopsies taken six months post-PDT treatment showed there were still residual viable cancer cells present in the prostate tissue sections. The authors propose that the incomplete treatment of the prostate tissue could be due to a too low light threshold dose, which was set to 5 J/cm2.